Methods and reagents for combined PCR amplification and hybridization probing

ABSTRACT

An oligonucleotide probe is disclosed, the probe including an oligonucleotide, a fluorescer molecule attached to a first end of the oligonucleotide and a quencher molecule attached to the opposite end of the oligonucleotide. The probe is rendered impervious to digestion by the 5′→3′ exonuclease activity of a polymerase and the 5′→3′ extension of by a polymerase. The invention also includes methods for performing combined PCR amplification and hybridization probing, one such method including the steps of contacting a target nucleic acid sequence with PCR reagents and an oligonucleotide probe as described above, and subjecting these reagents to thermal cycling. One preferred refinement of the above method further includes the addition of a strand displacer to facilitate amplification. Additional similar combined PCR hybridization methods are disclosed, such methods not requiring probes having their 5′ ends protected, wherein (i) the polymerase lacks 5′→3′ exonuclease activity, (ii) a 5′→3′ exonuclease inhibitor is included, and (iii) an exonuclease deactivation step is performed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/293,236, filed Nov. 12, 2002, now abandoned which is a continuationof U.S. patent application Ser. No. 08/826,538, filed Apr. 3, 1997, nowU.S. Pat. No. 6,485,903, which is a continuation of U.S. patentapplication Ser. No. 08/435,509, filed May 5, 1995, abandoned, all ofwhich are incorporated herein by reference.

BACKGROUND

This invention relates generally to the field of nucleic amplificationand probing, and more particularly, to methods and compositions forperforming PCR and probe hybridization using a single reagent mixture.

Nucleic acid amplification techniques provide powerful tools for thestudy of genetic material. The polymerase chain reaction (PCR) inparticular has become a tool of major importance finding applications incloning, analysis of genetic expression, DNA sequencing, geneticmapping, drug discovery, criminal forensics, and the like, e.g., Inniset al. in PCR Protocols A guide to Methods and Applications, AcademicPress, San Diego (1990); and U.S. Pat. Nos. 4,683,195, 4,683,202.

For many important applications, in addition to amplifying a targetnucleic acid sequence, it is desirable to further characterize suchsequence by treatment with a nucleic acid hybridization probe, i.e., alabeled single stranded polynucleotide which is complementary to all orpart of the target sequence, e.g., Nucleic Acid Hybridization, Hames etal. Eds., IRL Press, Oxford (1985). Probe hybridization may provideadditional sequence selectivity over simple PCR amplification as well asallowing for the characterization of multiple sequence sites within thetarget nucleic acid sequence in an independent manner.

Traditionally, PCR and probe hybridization processes have been performedas separate operations. However, it is highly desirable to perform boththe PCR and the probe hybridization reactions in a combined manner usinga single reagent mixture containing both PCR reagents and probingreagents. There are several advantages realized by combining the PCR andthe probing process: (i) only a single reagent addition step isrequired, thereby allowing the combined reactions to proceed withouthaving to open up the reaction tube, thereby reducing the opportunityfor sample mix-up and sample contamination; (ii) fewer reagents areneeded; (iii) fewer reagent addition steps are required, makingautomation more straight forward; and, (iv) in the case of in situmethods, there is no requirement to take apart a sample containmentassembly used to protect the integrity of the cellular sample duringthermocycling.

One existing method which combines PCR with hybridization probing in asingle reaction is the technique know as “Taqman”, e.g., Holland et al,Proc. Natl. Acad. Sci. USA 88: 7276–7280 (1991). In the Taqman assay, anoligonucleotide probe, nonextendable at the 3′ end, labeled at the 5′end, and designed to hybridize within the target sequence, is introducedinto the PCR reaction. Hybridization of the probe to a PCR reactionproduct strand during amplification generates a substrate suitable forthe exonuclease activity of the PCR polymerase. Thus, duringamplification, the 5′→3′ exonuclease activity of the polymerase enzymedegrades the probe into smaller fragments that can be differentiatedfrom undegraded probe. While a significant improvement over priormethods, the Taqman assay has a number of important drawbacks that limitits utility including (i) the requirement that the polymerase enzymeused for the PCR must include a 5′→3′ exonuclease activity, (ii) the5′→3′ exonuclease activity must be able to efficiently digest adye-labeled nucleotide, and (iii) the detectable product of thedigestion is a small, rapidly diffusable species which may impact theability to spatially locate the target sequence when applied to in situmethods.

A second existing method which combines PCR with probing in a singlereaction is that disclosed by Higuchi et al. in Biotechnology, 10:413–417 (1992). In this method, ethidium bromide is added to the PCRreaction and, since the fluorescence of the ethidium bromide increasesin the presence of double stranded DNA, an increase in fluorescence canbe correlated with the accumulation of double stranded PCR product.However, this method does not provide any sequence specificity beyondthe PCR reaction and is limited to the detection of a single sequencesite within the target nucleic acid sequence.

A third method which allows for combined amplification and probing stepsis that of Bagwell in EP 0601889A2. The probe in Bagwell's methodincludes a nucleotide sequence which has (i) a segment complementary tothe target nucleic acid and (ii) a segment capable of forming one ormore hairpins. The probe also includes covalently attached fluorescerand a quencher molecules located such that when a hairpin is formed, thefluorescer and quencher are in close enough proximity to allow resonanceenergy transfer between them. This method has the significant shortcoming that it limits the possible probe sequences to those capable offorming a hairpin structure. Moreover, the kinetics and thermodynamicsof probe-target binding will be unfavorably affected by the presence ofthe hairpin structure.

SUMMARY

The present invention relates generally to our discovery of methods andreagents useful for the combined amplification and hybridization probedetection of amplified nucleic acid target sequence in a single reactionvessel using a single reagent.

An object of our invention is to provide methods and reagents for theamplification and probe detection of amplified target sequences whereinthe amplification and probing steps are performed in a combined mannersuch that no reagent additions are required subsequent to theamplification step.

A further object of our invention is to provide methods and reagents forthe amplification and probe detection of amplified target sequenceslocated within cells or tissue sections wherein there is no need todisassemble a containment assembly between the amplification and probingsteps.

Another object of our invention is to provide methods and reagents forthe amplification and probe detection of amplified target sequenceswherein a single reagent mixture is used for both the amplification andprobing steps.

A further object of our invention is to provide methods and reagents forthe amplification and probing of amplified target sequences locatedwithin cells or tissue sections wherein no washing step is requiredbetween the amplification and probing steps.

Another object of our invention is to provide a probe composition foruse in the above methods that has detectabley different fluorescencecharacteristics depending on whether it is in a double stranded state,e.g., hybridized to a complementary target sequence, or whether it is ina single stranded state.

Yet another object of our invention is to provide oligonucleotide probeswhich are resistant to the 5′→3′ exonuclease activity of polymeraseenzymes.

Another object of our invention is to provide labeled probes in which,at the time of detection, the label is attached to a large, slowlydiffusing species, i.e., a species having a size greater than or equalto the size of the probe.

A further object of our invention is to provide probes which do notrequire hairpin structures in order to provide a differential signalbetween double stranded and single stranded states.

Another object of our invention is to provide methods and reagents forthe amplification and probe detection of amplified target sequenceswherein the polymerase is not required to have 5′→3′ exonucleaseactivity.

Yet another object of our invention is to provide methods and reagentsfor the amplification and probe detection of amplified target sequenceswherein multiple sequence sites can be detected within a single targetsequence.

Still another object of our invention is to provide various reagent kitsuseful for the practice of the aforementioned methods.

The foregoing and other objects of the invention are achieved by, in oneaspect, an oligonucleotide probe which is made up of an oligonucleotidecapable of hybridizing to a target polynucleotide sequence. Theoligonucleotide is modified such that the 5′ end is rendered imperviousto digestion by the 5′→3′ exonuclease activity of a polymerase, and the3′ end is rendered impervious to the 5′→3′ extension activity of apolymerase. Furthermore, the oligonucleotide probe includes a fluorescermolecule attached to a first end of the oligonucleotide, and a quenchermolecule attached to a second end of the oligonucleotide such that thequencher molecule substantially quenches the fluorescence of thefluorescer molecule whenever the oligonucleotide probe is in asingle-stranded state and such that the fluorescer is substantiallyunquenched whenever the oligonucleotide probe is in a double-strandedstate. Alternatively, the fluorescer and quencher are separated by atleast 18 nucleotides.

In a second aspect, the invention provides a first method for performingcombined PCR amplification and hybridization probing. In the method, atarget nucleic acid sequence is contacted with PCR reagents, includingat least two PCR primers, a polymerase enzyme, and an oligonucleotideprobe of the invention as described above. This mixture is thensubjected to thermal cycling, the thermal cycling being sufficient toamplify the target nucleic acid sequence specified by the PCR reagents.

In a third aspect, the invention provides a second method for performingcombined PCR amplification and hybridization probing wherein the targetnucleic acid sequence is contacted with PCR reagents, including at leasttwo PCR primers and a polymerase enzyme substantially lacking any 5′→3′exonuclease activity, and an oligonucleotide probe. The oligonucleotideprobe includes a fluorescer molecule attached to a first end of theoligonucleotide and a quencher molecule attached to a second end of theoligonucleotide such that quencher molecule substantially quenches thefluorescence of the fluorescer molecule whenever the oligonucleotideprobe is in a single-stranded state and such that the fluorescer issubstantially unquenched whenever the oligonucleotide probe is in adouble-stranded state. In addition, the 3′ end of the probe is renderedimpervious to the 5′→3′ extension activity of a polymerase. The targetnucleic acid sequence, the oligonucleotide probe, and the PCR reagentsare subjected to thermal cycling sufficient to amplify the targetnucleic acid sequence specified by the PCR reagents.

In one preferred embodiment, rather than requiring the polymerase enzymeto be lacking any 5′→3′ exonuclease activity, an exonuclease activityinhibitor is added to the reaction, the inhibitor being sufficient toinhibit the 5′→3′ exonuclease activity of the polymerase at a probehybridization temperature.

In a second preferred embodiment, rather than requiring the polymeraseenzyme to be lacking any 3′→5′ exonuclease activity, or rather thanadding an exonuclease activity inhibitor, the 3′→5′ exonuclease activityof the polymerase is deactivated prior to detecting the probe.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of a first preferred combined PCR andprobe hybridization method wherein a temperature difference between themelting temperature (T_(m)) of the probe and the reaction temperature ofthe PCR polymerization step is used to prevent the probe frominterfering with the PCR polymerization step and digestion of the probe.

FIG. 2 shows a schematic diagram of a second preferred combined PCR andprobe hybridization method wherein a strand displacer is used to preventthe probe from interfering with the PCR polymerization step anddigestion of the probe.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference will now be made in detail to the preferred embodiments of theinvention. While the invention will be described in conjunction with thepreferred embodiments, it will be understood that they are not intendedto limit the invention to those embodiments. On the contrary, theinvention is intended to cover alternatives, modifications, andequivalents which may be included within the invention as defined by theappended claims.

1. PCR and In Situ PCR

As used herein, the term “PCR reagents” refers to the chemicals, apartfrom the target nucleic acid sequence, needed to perform the PCRprocess. These chemicals generally consist of five classes ofcomponents: (i) an aqueous buffer, (ii) a water soluble magnesium salt,(iii) at least four deoxyribonucleotide triphosphates (dNTPs), (iv)oligonucleotide primers (normally two primers for each target sequence,the sequences defining the 5′ ends of the two complementary strands ofthe double-stranded target sequence), and (v) a polynucleotidepolymerase, preferably a DNA polymerase, more preferably a thermostableDNA polymerase, i.e., a DNA polymerase which can tolerate temperaturesbetween 90° C. and 100° C. for a total time of at least 10 min withoutlosing more than about half its activity.

The four conventional dNTPs are thymidine triphosphate (dTTP),deoxyadenosine triphosphate (dATP), deoxycitidine triphosphate (dCTP)and deoxyguanosine triphosphate (dGTP). These conventional triphosphatesmay be supplemented or replaced by dNTPs containing base analogues whichWatson-Crick base pair like the conventional four bases, e.g.,deoxyuridine triphosphate (dUTP).

“In situ PCR” refers to PCR amplification performed in fixed cells, suchthat specific amplified nucleic acid is substantially contained within acell or subcellular structure which originally contained the targetnucleic acid sequence. The cells may be in aqueous suspension or may bepart of a tissue sample, e.g., histochemical section, or a cytochemicalsmear. As used herein, the term “histochemical section” refers to asolid sample of biological tissue which has been frozen or chemicallyfixed and hardened by embedding in a wax or plastic, sliced into a thinsheet (typically several microns thick), and attached to a solidsupport, e.g., a microscope slide, and the term “cytochemical smear”refers to a suspension of cells, e.g., blood cells, which has beenchemically fixed and attached to a microscope slide. Preferably, thecells will have been rendered permeable to PCR reagents by proteinasedigestion, by liquid extraction with a surfactant or organic solvent, orother like permeablization methods.

As used herein, the term “fixed cells” refers to a sample of biologicalcells which has been chemically treated to strengthen cellularstructures, particularly membranes, against disruption by solventchanges, temperature changes, mechanical stress or drying. Cells may befixed either in suspension our as part of a tissue sample. Cellfixatives generally are chemicals which crosslink the proteinconstituents of cellular structures, most commonly by reacting withprotein amino groups. Preferred fixatives include buffered formalin, 95%ethanol, fomaldehyde, paraformaldehyde, and glutaraldehyde. Thepermeability of fixed cells can be increased by treatment withproteinases, or with surfactants or organic solvents which dissolvemembrane lipids.

2. Oligonucleotide Probes

The term “oligonucleotide” as used herein includes linear oligomers ofnatural or modified monomers or linkages, includingdeoxyribonucleotides, ribonucleotides, and the like, capable ofspecifically binding to a target polynucleotide by way of a regularpattern of monomer-to-monomer interactions, such as Watson-Crick type ofbase pairing, or the like. Usually monomers are linked by phosphodiesterbonds or analogs thereof to form oligonucleotides ranging in size from afew monomeric units, e.g. 3–4, to several tens of monomeric units.Whenever an oligonucleotide is represented by a sequence of letters,such as “ATGCCTG,” it will be understood that the nucleotides are in5′→3′ order from left to right and that “A” denotes deoxyadenosine, “C”denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotesthymidine, unless otherwise noted. Analogs of phosphodiester linkagesinclude phosphorothioate, phosphoranilidate, phosphoramidate, and thelike.

As used herein, “nucleotide” includes the natural nucleotides, including2′-deoxy and 2′-hydroxyl forms, e.g. as described in Kornberg and Baker,DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). “Analogs” inreference to nucleotides includes synthetic nucleotides having modifiedbase moieties and/or modified sugar moieties, e.g. described by Scheit,Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman,Chemical Reviews, 90: 543–584 (1990), or the like, with the only provisothat they are capable of specific hybridization. Such analogs includesynthetic nucleotides designed to enhance binding properties, reducedegeneracy, increase specificity, reduce activity as enzyme substrates,and the like.

Oligonucleotides of the invention can be synthesized by a number ofapproaches, e.g. Ozaki et al, Nucleic Acids Research, 20: 5205–5214(1992); Agrawal et al, Nucleic Acids Research, 18: 5419–5423 (1990); orthe like. The oligonucleotide probes of the invention are convenientlysynthesized on an automated DNA synthesizer, e.g. a Perkin-Elmer (FosterCity, Calif.) Model 392 or 394 DNA/RNA Synthesizer, using standardchemistries, such as phosphoramidite chemistry, e.g. disclosed in thefollowing references: Beaucage and Iyer, Tetrahedron, 48: 2223–2311(1992); Molko et al, U.S. Pat. No. 4,980,460; Koster et al, U.S. Pat.No. 4,725,677; Caruthers et al, U.S. Pat. Nos. 4,415,732; 4,458,066; and4,973,679; and the like. Alternative chemistries, e.g. resulting innon-natural backbone groups, such as phosphorothioate, phosphoramidate,and the like, may also be employed provided that the hybridizationefficiencies of the resulting oligonucleotides are not adverselyaffected. Preferably, the oligonucleotide probe is in the range of15–150 nucleotides in length. More preferably, the oligonucleotide probeis in the range of 18–30 nucleotides in length. The precise sequence andlength of an oligonucleotide probe of the invention depends in part onthe nature of the target nucleic acid sequence to which it hybridizes.The binding location and length may be varied to achieve appropriateannealing and melting properties for a particular embodiment. Guidancefor making such design choices can be found in many of the above-citedreferences describing the “Taqman” type of assays.

Preferably, the 3′ terminal nucleotide of the oligonucleotide probe isrendered incapable of extension by a nucleic acid polymerase. Suchblocking may be carried out by the attachment of a fluorescer orquencher molecule to the terminal 3′ carbon of the oligonucleotide probeby a linking moiety, or by making the 3′-terminal nucleotide adideoxynucleotide. Alternatively, the 3′ end of the oligonucleotideprobe is rendered impervious to the 5′→3′ extension activity of apolymerase by including one or more modified internucleotide linkagesinto the 3′ end of the oligonucleotide. Minimally, the 3′-terminalinternucleotide linkage must be modified, however, up to all theinternucleotide linkages may be modified. Such internucleotidemodifications may include phosphorothioate linkeages, e.g.,Oligonucleotides and Analogs, Chaps. 4 and 5, IRL Press, New York(1991); methylyphosphonate linkages, Oligonucleotides and Analogs, Chap.6, IRL Press, New York (1991); boranophosphate linkages, e.g., Shaw etal., Methods Mol. Biol. 20: 225–43 (1993); and other like polymeraseresistant internucleotide linkages. An alternative method to block 3′extension of the probe is to form an adduct at the 3′ end of the probeusing mitomycin C or other like antitumor antibiotics, e.g., Basu etal., Biochemistry, 32: 4708–4718 (1993).

In an important aspect of one embodiment of the present invention, theoligonucleotide probe is rendered impervious to degradation by the 5′→3′exonuclease activity of a nucleic acid polymerase. Preferably, the 5′end of the oligonucleotide probe is rendered impervious to digestion byincluding one or more modified internucleotide linkages into the 5′ endof the oligonucleotide. Minimally, the 5′-terminal internucleotidelinkage must be modified, however, up to all the internucleotidelinkages in the oligonucleotide may be modified. Such internucleotidemodifications may include modified linkages of the type used in thesynthesis of anti-sense oligonucleotides. Examples of such nucleaseresistant linkages include phosphorothioate linkages, e.g.,Oligonucleotides and Analogs, Chaps. 4 and 5, IRL Press, New York(1991); methylyphosphonate linkages, Oligonucleotides and Analogs, Chap.6, IRL Press, New York (1991); boranophosphate linkages, e.g., Shaw etal., Methods Mol. Biol. 20: 225–43 (1993); polyamide nucleic acid (PNA)linkages, e.g., Nielsen et al., Science, 254: 1497–1500 (1991), andother like exonuclease resistant linkages. Alternatively, a peptidemolecule is be attached to the 5′ end of the probe in an manneranaologus to that of Soukchareun et al. in Bioconjugate Chemistry, 6:43–53 (1995).

In another important aspect of the oligonucleotide probes of the presentinvention, the probes include fluorescer and quencher molecules attachedto the oligonucleotide. As used herein, the terms “quenching” or“fluorescence energy transfer” refer to the process whereby when afluorescer molecule and a quencher molecule are in close proximity,whenever the fluorescer molecule is excited, a substantial portion ofthe energy of the excited state nonradiatively transfers to the quencherwhere it either dissipates nonradiatively or is emitted at a differentemission wavelength than that of the fluorescer.

It is well known that the efficiency of quenching is a strong functionof the proximity of the fluorescer and the quencher, i.e., as the twomolecules get closer, the quenching efficiency increases. As quenchingis strongly dependent on the physical proximity of the reporter moleculeand quencher molecule, it has been assumed that the quencher andreporter molecules must be attached to the probe within a fewnucleotides of one another, usually with a separation of about 6–16nucleotides, e.g. Lee et al. Nucleic Acids Research, 21: 3761–3766(1993); Mergny et al, Nucleic Acids Research, 22: 920–928 (1994);Cardullo et al, Proc. Natl. Acad. Sci., 85: 8790–8794 (1988); Clegg etal, Proc. Natl. Acad. Sci., 90: 2994–2998 (1993); Ozaki et al, NucleicAcids Research, 20: 5205–5214 (1992); and the like. Typically, thisseparation is achieved by attaching one member of a reporter-quencherpair to the 5′ end of the probe and the other member to a base 6–16nucleotides away.

However, it has been recognized as part of the present invention that byplacing the fluorescer and quencher molecules at seemingly remotelocations on the oligonucleotide, differential quenching can be seenbetween the single stranded state and the double stranded state, i.e.,hybridized state, of the oligonucleotide probe, e.g., Bagwell et al.,Nucleic Acids Research, 22(12): 2424–2425 (1994); Bagwell, EP 0 601 889A2. The fluorescence signals can differ by as much as a factor of 20between the single stranded and double stranded states when thefluorescer and quencher are separated by 20 bases. This effect is mostprobably due to the fact that in the single stranded state, theoligonucleotide exists as a flexible random coil structure which allowsthe ends of the oligonucleotide to be in close proximity, while, in thedouble stranded state, the oligonucleotide exists as a rigid, extendedstructure which separates the fluorescer and quencher. Thus, using thisarrangement, one sees relatively efficient quenching of the fluorescerwhen the oligonucleotide probe is in the single stranded or unhybridizedstate and relatively inefficient quenching of the fluorescer when theoligonucleotide probe is in the double stranded or hybridized state.

Preferably, fluorescer molecules are fluorescent organic dyesderivatized for attachment to the terminal 3′ carbon or terminal 5′carbon of the probe via a linking moiety. Preferably, quencher moleculesare also organic dyes, which may or may not be fluorescent, depending onthe embodiment of the invention. For example, in a preferred embodimentof the invention, the quencher molecule is fluorescent. Generally,whether the quencher molecule is fluorescent or simply releases thetransferred energy from the fluorescer by non-radiative decay, theabsorption band of the quencher should substantially overlap thefluorescent emission band of the fluorescer molecule. Non-fluorescentquencher molecules that absorb energy from excited fluorescer molecules,but which do not release the energy radiatively, are referred to hereinas chromogenic molecules.

There is a great deal of practical guidance available in the literaturefor selecting appropriate fluorescer-quencher pairs for particularprobes, as exemplified by the following references: Clegg (cited above);Wu et al., Anal. Biochem., 218: 1–13 (1994). Pesce et al, editors,Fluorescence Spectroscopy (Marcel Dekker, New York, 1971); White et al,Fluorescence Analysis: A Practical Approach (Marcel Dekker, New York,1970); and the like. The literature also includes references providingexhaustive lists of fluorescent and chromogenic molecules and theirrelevant optical properties for choosing fluorescer-quencher pairs, e.g.Berlman, Handbook of Fluorescence Sprectra of Aromatic Molecules, 2ndEdition (Academic Press, New York, 1971); Griffiths, Colour andConsitution of Organic Molecules (Academic Press, New York, 1976);Bishop, editor, Indicators (Pergamon Press, Oxford, 1972); Haugland,Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes,Eugene, 1992); Pringsheim, Fluorescence and Phosphorescence(Interscience Publishers, New York, 1949); and the like. Further, thereis extensive guidance in the literature for derivatizing fluorescer andquencher molecules for covalent attachment via common reactive groupsthat can be added to an oligonucleotide, as exemplified by the followingreferences: Haugland (cited above); Ullman et al, U.S. Pat. No.3,996,345; Khanna et al, U.S. Pat. No. 4,351,760; and the like.

Exemplary fluorescer-quencher pairs may be selected from xanthene dyes,including fluoresceins, and rhodamine dyes. Many suitable forms of thesecompounds are widely available commercially with substituents on theirphenyl moieties which can be used as the site for bonding or as thebonding functionality for attachment to an oligonucleotide. Anothergroup of fluorescent compounds are the naphthylamines, having an aminogroup in the alpha or beta position. Included among such naphthylaminocompounds are 1-dimethylaminonaphthyl-5-sulfonate,1-anilino-8-naphthalene sulfonate and 2-p-touidinyl-6-naphthalenesulfonate. Other dyes include 3-phenyl-7-isocyanatocoumarin, acridines,such as 9-isothiocyanatoacridine and acridine orange;N-(p-(2-benzoxazolyl)phenyl)maleimide; benzoxadiazoles, stilbenes,pyrenes, and the like.

Preferably, fluorescer and quencher molecules are selected fromfluorescein and rhodamine dyes. These dyes and appropriate linkingmethodologies for attachment to oligonucleotides are described in manyreferences, e.g. Khanna et al (cited above); Marshall, Histochemical J.,7: 299–303 (1975); Mechnen et al, U.S. Pat. No. 5,188,934; Menchen etal, European patent application 87310256.0; and Bergot et al,International application PCT/US90/05565. The latter four documents arehereby incorporated by reference.

There are many linking moieties and methodologies for attachingfluorescer or quencher molecules to the 5′ or 3′ termini ofoligonucleotides, as exemplified by the following references: Eckstein,editor, Oligonucleotides and Analogues: A Practical Approach (IRL Press,Oxford, 1991); Zuckerman et al, Nucleic Acids Research, 15: 5305–5321(1987)(3′ thiol group on oligonucleotide); Sharma et al, Nucleic AcidsResearch, 19: 3019 (1991)(3′ sulfhydryl); Giusti et al, PCR Methods andApplications, 2: 223–227 (1993) and Fung et al, U.S. Pat. No. 4,757,141(5′ phosphoamino group via Aminolink™ II available from AppliedBiosystems, Foster City, Calif.); Stabinsky, U.S. Pat. No. 4,739,044 (3′aminoalkylphosphoryl group); Agrawal et al, Tetrahedron Letters, 31:1543–1546 (1990)(attachment via phosphoramidate linkages); Sproat et al,Nucleic Acids Research, 15: 4837 (1987)(5′ mercapto group); Nelson etal, Nucleic Acids Research, 17: 7187–7194 (1989)(3′ amino group); andthe like.

Preferably, commercially available linking moieties are employed thatcan be attached to an oligonucleotide during synthesis, e.g. availablefrom Clontech Laboratories (Palo Alto, Calif.).

Rhodamine and fluorescein dyes are also conveniently attached to the 5′hydroxyl of an oligonucleotide at the conclusion of solid phasesynthesis by way of dyes derivatized with a phosphoramidite moiety, e.g.Woo et al, U.S. Pat. No. 5,231,191; and Hobbs, Jr. U.S. Pat. No.4,997,928.

3. Combined PCR Amplification and Probe Hybridization

There are three key issues which must be addressed when performingcombined PCR and probe hybridization assays: (i) the oligonucleotideprobe should not block or otherwise interfere with the PCRpolymerization step thereby reducing the stepwise efficiency of theamplification, where as used herein, the term “polymerization step”refers to the step in the PCR process in which the primers are extendedfrom their 3′ ends by incorporation of nucleotide bases by apolymerase-mediated reaction; (ii) the oligonucleotide probe must not bedigested by the 5′→3′ exonuclease activity of the polymerase enzyme; and(iii) the probe should be incapable of 5′→3′ extension by thepolymerase.

In one preferred embodiment of the present invention, the probe isprotected from interfering with the PCR polymerization step by designingthe probe such that its melting temperature is below the temperature ofthe PCR polymerization step. As used herein, the term “meltingtemperature” is defined as a temperature at which half of the probe ishybridized to a target sequence, i.e., in a double stranded state, andhalf is in unhybridized, i.e., in a single stranded state. Preferably,the melting temperature of the probe is between 40° C. and 55° C., andthe melting temperature of the PCR primers is between 55° C. and 70° C.

Referring to FIG. 1, during the PCR polymerization step, the probe isunhybridized, i.e., in a single stranded state, and thereby quenched.Moreover, because the probe is not bound to the target sequence, the PCRpolymerization can proceed without interference from the probe. Next,during the hybridization step, the temperature is reduced to ahybridization temperature, preferably a temperature at or below theT_(m) of the probe, causing the probe to hybridize to the target. Thehybridization of the probe causes a reduction in the amount ofquenching, resulting in a measurable signal which is indicative of probehybridization to the target sequence, such signal also providingquantitative information as to the mount of target sequence present.During the hybridization step, the probe will not be digested by theexonuclease activity of the polymerase because, as discussed above, theprobe has been designed to be impervious to the 5′→3′ exonucleaseactivity of the polymerase.

In a variation on the above described T_(m) mediated combined PCR andprobe hybridization method, rather than using a probe which isimpervious to the 5′→3′ exonuclease activity of the polymerase,digestion of the probe during the hybridization step is prevented byusing a polymerase which lacks a 5′→3′ exonuclease activity. Examples ofsuch 5′→3′ minus polymerases include DNA polymerase I Klenow fragment,T4 DNA polymerase, and T7 DNA polymerase, and other like 5′→3′exonuclease minus DNA polymerases, e.g., Amershan Life Science, Inc.,Arlington Heights, Ill.

In a second variation of the above described T_(m) mediated combined PCRand probe hybridization method, rather than using a probe which isimpervious to the 5′→3′ exonuclease activity of the polymerase or usingan exonuclease-minus polymerase to protect the probe from exonucleasedigestion, the polymerase is rendered inactive with respect to itsexonuclease activity during the hybridization step. Such inactivationcan be achieved in a number of ways including (i) introducing atemperature sensitive inhibitor into the reaction which will inhibit the5′→3′ exonuclease activity of the polymerase at the hybridizationtemperature, e.g., a solid adsorbent, a specific antibody molecule, orother like reversible or irreversible polymerase inhibitors; (ii) usinga polymerase whose activity is greatly reduced at the hybridizationtemperature; or (iii) introducing an enzyme deactivation step prior tothe hybridization step which irreversibly kills the polymerase enzyme,i.e., an extended period at high temperature.

In a second preferred embodiment of the present invention, the probe isprevented from interfering with the PCR polymerization step by includinga strand displacer into the reaction, where, as used herein, the term“strand displacer” refers to an agent capable of causing thedisplacement of an oligonucleotide probe from a target to which it ishybridized, e.g., a DNA helicase, e.g., Howard et al., Proc. Natl. Acad.Sci. USA 91: 12031–12035 (1994), or a single-stranded binding protein,E. G. Zijderveld, Journal of Virology 68(2): 1158–1164 (1994). Referringto FIG. 2, in this embodiment, during the polymerization step, thestrand displacer displaces the probe from the template strand therebyallowing the polymerization step to proceed without interference fromthe probe. Then, in order to allow hybridization of the probe during thehybridization step, the strand displacer is rendered inactive. Suchinactivation can be achieved in a number of ways including thosedescribed above with reference to inactivation of exonuclease activity.Generally, the strand displacement activity of a strand displacer ishigher for longer oligonucleotide duplexes, thus, the PCR primersthemselves are not susceptible to strand displacement during the PCRreaction.

4. EXAMPLES

The Invention will be further clarified by a consideration of thefollowing examples, which are intended to be purely exemplary of theinvention.

Example 1

Comparison of Fluorescence Emissions of Probes in Single Stranded andDouble Stranded States

Linker arm nucleotide (“LAN”) phosphoramidite was obtained from GlenResearch. Standard DNA phosphoramidites, 6-carboxyfluorescein (“6-FAM”)phosphoramidite, 6-carboxytetramethylrhodamine succinimidyl ester(“TAMRA NHS ester”), and Phosphalink™ for attaching a 3′ blockingphosphate were obtained from Perkin-Elmer, Applied Biosystems Division.Oligonucleotide synthesis was performed on a model 394 DNA Synthesizer(Applied Biosystems). Oligonucleotides were purified using OligoPurification Cartridges (Applied Biosystems).

Doubly labeled probes were synthesized with 6-FAM-labeledphosphoramidite at the 5′ end, LAN replacing one of the T's in theoligonucleotide sequence, and Phosphalink™ at the 3′ end. Followingdeprotection and ethanol precipitation, TAMRA NHS ester was coupled tothe LAN-containing oligonucleotide in 250 mM Na-bicarbonate buffer (pH9.0) at room temperature. Unreacted dye was removed by passage over aPD-10 Sephadex column. Finally, the doubly labeled probe was purified bypreparative HPLC using standard protocols.

The oligonucleotide sequences of the probes and their complements areshown in Table 1. As used herein, the term “complement” refers to anoligonucleotide sequence which is capable of hybridizing specificallywith a probe sequence.

TABLE 1 Probe/ Complement Sequence 1 ATGCCCTCCCCCATGCCATCCT (SEQ IDNO:1) GCGT 1 (Complement) AGACGCAGGATGGCATGGGGGA (SEQ ID NO:2) GGGCATAC2 CGCCCTGGACTTCGAGCAAGAG (SEQ ID NO:3) AT 2 (Complement)CCATCTCTTGCTCGAAGTCCAG (SEQ ID NO:4) GGCGAC 3 TCGCATTACTGATCGTTGCCAA(SEQ ID NO:5) CCAGT 3 (Complement) GTACTGGTTGGCAACGATCAGT (SEQ ID NO:6)AATGCGATG 4 CGGATTTGCTGGTATCTATGAC (SEQ ID NO:7) AAGGAT 4 (Complement)TTCATCCTTGTCATAGATACCA (SEQ ID NO:8) GCAAATCCG

Four pairs of probes were studied. For each pair, one probe has TAMRAattached to an internal nucleotide, the other has TAMRA attached to the3′ end nucleotide, and both probes have 6-FAM attached to the 5′ end. Tomeasure the fluorescence of the probes in a single stranded state,fluorescence emissions at 518 nm were measured using solutionscontaining a final concentration of 50 nM probe, 10 mM Tris-HCl (pH8.3), 50 mM KCl, and 10 mM MgCl₂. To measure the fluorescence of theprobes in a double stranded state, the solutions additionally contained100 mM complement oligonucleotide. Before addition of the MgCl₂, 120 μlof each sample was heated at 95° C. for 5 min. Following the addition of80 μl of 25 mm MgCl₂, each sample was allowed to cool to roomtemperature and the fluorescence emissions were measured. Reportedvalues are the average of three measurements. Table 2 gives the resultsof fluorescence measurements of the indicated probes in single anddouble stranded states. As can be seen from the data in Table 2, forprobes having the fluorescer and quencher at opposite ends of theoligonucleotide, hybridization caused a dramatic increase in the degreeof differential quenching over that seen when the fluorescer andquencher were closer together. For longer probes, we would expect thatthere exists an optimum separation between the fluorescer and thequencher such that rather than placing the fluorescer and quencher atoppisite ends, they are both located internally but separated by someoptimum distance.

TABLE 2 TAMRA Differential Probe Location^(a) Quenching^(b) 1 7 2.5 1 2611.8 2 6 3.7 2 24 19.2 3 7 2.0 3 27 8.0 4 10 5.3 ^(a)The TAMRA locationis expressed as the number of nucleotides from the 5′ end of theoligonucleotide in a 5′ to 3′ direction. ^(b)Differential quenching isdefined as the fluorescence emission intensity of the probe in thedouble stranded state divided by the fluorescence emission intensity ofthe probe in the single stranded state.

Although only a few embodiments have been described in detail above,those having ordinary skill in the molecular biology art will clearlyunderstand that many modifications and variations are possible in thepreferred embodiments without departing from the teachings thereof.

1. A method for performing combined PCR amplification and hybridizationprobing comprising the steps of: contacting a target nucleic acidsequence with PCR reagents, including at least two PCR primers and apolymerase enzyme, and an oligonucleotide probe comprising: anoligonucleotide capable of hybridizing to a target polynucleotidesequence; a fluorescer molecule attached to a first location on theoligonucleotide; a quencher molecule attached to a second location onthe oligonucleotide such that the quencher molecule substantiallyquenches the fluorescence of the fluorescer molecule whenever theoligonucleotide probe is not hybridized to the target polynucleotidesequence and such that the fluorescer molecule is substantiallyunquenched whenever the oligonucleotide probe is hybridized to thetarget polynucleotide sequence; a 5′ end which is not recognized by apolymerase having a 5′→3′ exonuclease activity; and a 3′ end which isnot recognized by a polymerase having a 5′→3′ extension activity; andsubjecting the target nucleic sequence, the oligonucleotide probe, andthe PCR reagents to thermal cycling, including a polymerization step,the thermal cycling being sufficient to amplify the target nucleic acidsequence specified by the PCR reagents.
 2. The method of claim 1 whereinsaid fluorescer molecule is a flourescein dye and said quencher moleculeis a rhodamine dye.
 3. The method of claim 2 wherein said first locationon said oligonucleotide is the 5′ end.
 4. The method of claim 1 furthercomprising the step of measuring the extent of fluorescence quenching ofthe oligonucleotide probe, such measurement being performed subsequentto thermocycling and at a probe hybridization temperature.
 5. The methodof claim 1 wherein the target nucleic acid sequence is located withinone or more fixed cells.
 6. The method of claim 5 further comprising thestep of measuring the extent of fluorscence quenching of theoligonucleotide probe at a hybridization temperature in a manner whichlocates the probe within the individual cells originally containing thetarget nucleic acid sequence.
 7. The method of claim 5 wherein the fixedcells, the PCR reagents,and the oligonucleotide probe are located in acontainment assembly.
 8. The method of claim 1 wherein the probehybridization temperature is less than or equal to the temperature ofthe polymerization step of the thermocycling.
 9. The method of claim 1further comprising the step of adding a strand displacer prior tothermal cycling for preventing the oligonucleotide probe from blockingthe 5′—>3′ extension of an upstream PCR primer during the polymerizationstep.
 10. The method of claim 9 wherein the strand displacer is ahelicase.
 11. The method of claim 1 wherein the fluorescer and quencherare separated by at least 18 nucleotides.